While living systems are often viewed as the reaction of biochemicals in water, considerable research has been devoted to the study of the structure and function of biochemicals in mixed water-organic solvent systems. In 1969, Kaminsky and Davidson (1) (see bibliography concluding this section for citations to referenced publications) examined the effects of organic solvents on the low molecular weight protein cytochrome c (12K daltons). A similar approach was used by A. A. Klyosov et al. (2) and Bettelheim and Lukton (3) to measure the enzymatic activity of small molecular weight proteins.
Gitler and Montal (4) radically departed from these early studies in their stabilization of cytochrome c in a solvent system composed primarily (90%) of a water-immiscible solvent, decane, by complexation with a biological detergent, i.e., phospholipids. Marinek et al. (5) explored the stabilization and subsequent enzymatic activity of another low molecular weight protein, chymotrypsin (22K daltons), in iso-octane. The anionic detergent used by Marinek et al. was a non-biological mimic of phospholipids, namely sodium bis-(2-ethylhexyl)-sulfosuccinate ("AOT"). Similarly, Luisi et al. (6) found that chymotrypsin, and other low molecular weight enzymes, trypsin and pepsin (23K and 35K daltons, respectively), retained their enzymatic activity in the water-immiscible solvent cyclohexane when complexed with the cationic detergent methyltrioctylammonium chloride. Luisi et al. suggested that those enzymes retained their basic natural structure within a small aqueous core surrounded by a sphere of detergent and organic solvent. Such structures were termed "reverse micelles", forming within water-immiscible solvents when detergent content is above the critical micelle concentration ("CRC") and when the molar ratio of water to detergent ("W.sub.o ") is in the range from 5:1 to 50:1, inclusive. The bulk of these solutions (80-90%) is composed of a water-immiscible organic solvent.
Physical evidence tending to confirm the existence of reverse micelles has been gained from quasi-elastic light scattering, Huang and Kim (7), and small-angle neutron scattering measurements, M. Kotlarchyk et al. (8). Structural and mechanistic information concerning proteins within reverse micelles has been obtained through NMR (DeMarco et al. (9)), ultraviolet, circular dichroism and fluorescence (P. L. Luisi, 1979 (6)) spectroscopies, as well as enzymatic kinetic analyses.
A variety of low molecular weight proteins and enzymes have been stabilized in a variety of solvents and with a variety of surfactant and co-surfactant systems (P. L. Luisi, 1985 (10)). Although a diverse set of conditions was used in these experiments, some conclusions can be drawn. First, these studies were largely performed with relatively low molecular weight proteins (e.g., cytochrome c (12K daltons), ribonuclease (13K daltons), lysozyme (14K daltons), chymotrypsin (23K daltons), trypsin (35K daltons), alcohol dehydrogenase (37K daltons), rhodopsin (38K daltons), and lipase (45K daltons)), when compared to the molecular weight of the immunoglobulin molecules (150K daltons). Second, the majority of these proteins displays maximal catalytic activity at a water-to-detergent ratio (W.sub.o) of about 10-15. (We note that lipase of 100K daltons has been reported, and that this enzyme has been said to have an activity which increases up to W.sub.o of about 30 and then levels off with (Luisi et al. (12)); however, the molecular weight of this enzyme is significantly less than that of an antibody as contemplated by the present invention, and further the enzyme is lipophilic rather than hydrophilic, and thus its behavior should not be viewed as indicative of antibody behavior. And finally, as suggested by P. L. Luisi et al. (6), "Another interesting property of the phase transfer process . . . is its selectivity towards certain proteins" (p. 751). Factors such as protein size and charge, solution pH, surfactant composition and concentration as well as ionic strength, determine if a particular type of protein can be successfully stabilized within reverse micelles. Particular combinations of these factors are specific to different types of proteins, and this undercuts the predictability of whether one protein type can be stabilized from the finding that a different protein type can. Accordingly, it would not have logically followed that the teachings of the aforementioned workers could be extrapolated beyond the specific conditions and materials utilized by them.
It is also important to note that while stabilization of enzymes within water/water-immiscible biphasic systems was explored to catalyze reactions with water-insoluble substrates (Larsson (11); P. L. Luisi et al. (12)), or for mimicking physiological conditions (Han and Rhee (13)), there are other applications of reverse micelles such as both liquid and HPLC chromatography (M. A. Herandez, 1986 (14)), with supercritical fluids (R. W. Gale et al., 1987 (15)) and in protein extraction (Goklen and Hatton, 1985 (16)).
However, reports of antibody stabilization in these water-immiscible solvents (both organic liquids and supercritical fluids) are extremely limited.
Karr et al. (17) examined a covalent modification of IgG molecules with polyethylene glycol (PEG) as a method of improving the solubility of antibodies in a dextran phase of a biphasic solution. While a dextran phase is far more water-miscible than iso-octane or benzene, the major drawback of this approach is the extra time-consuming steps required for forming the PEG-antibody covalent complex and the possibility that chemical modification of a protein with PEG could also covalently modify amino acid residues in the antigen binding site, thereby reducing the antibody's antigen-binding capacity. In addition, there is no guarantee that this technique works with water-immiscible solvents.
Eremin et al. (18) examined the effects of antibodies directed against horseradish peroxidase in 50 mM aqueous AOT solutions in heptane. The thrust of this work was to study the loss of peroxidase activity observed due to mixing of enzyme and polyclonal anti-peroxidase antibodies. But there is no indication that the antibody was of a monoclonal nature or had a molecular weight which would have been conducive to solubilization in the reverse micelles at the conditions utilized, that the enzyme was inactivated by the antibody, or even that the system of Eremin et al. contained an effective amount of antibody in a functional state. Significantly, no attempt was made to test the antibody's antigen-binding capability in an ELISA analysis after solubilization in iso-octane. Since the presence of large antibodies in the polyclonal mixture, such as those of the IgM nature (970K daltons), could well have caused the applied conditions to be unfavorable for solubilization of antibodies or horseradish peroxidase, this article would not have disclosed use of an effective amount of functional antibodies in an aqueous/non-aqueous biphasic system to the skilled worker.
The development of information concerning conditions for stabilizing an effective amount of antibody or antibody fragment in a heterogeneous system comprising a dispersed aqueous phase, with retention of an effective amount of antibody or antibody fragment in a functional state, would be a significant step forward in the art.
(1) L. S. Kaminsky and A. J. Davidson, FEBS Letters 3, 338 (1969). PA0 (2) A. A. Klyosov, N. Van Viet, and I. V. Berezin, Eur. J. Biochem. 59, 3 (1975). PA0 (3) F. A. Bettelheim and A. Lukton, Nature 198, 357 (1963). PA0 (4) (a) C. Gitler and M. Montal, Biochem and Biophys Res. Comm. 47, 1486 (1972); (b) FEBS Letters 28, 329 (1972). PA0 (5) K. Martinek, A. V. Levashoft, N. L. Klyachko, and I. V. Berezin, Doklad Ada Nauk SSSR (Engl. edit), 236, 951 (1978). PA0 (6) P. L. Luisi, F. J. Bonner, A. Pellegrini, P. Wiget, and R. Wolf, Helv. Chim. Acta 62, 740 (1979). PA0 (7) J. S. Huang and M. W. Kim, Phys. Rev. Lett. 47, 1462 (1981). PA0 (8) M. Kotlarchyk, S. -H. Chen, J. S. Huang, and M. W. Kim, Phys. Rev. A 29, 2054 (1984). PA0 (9) A. De Marco, E. Menegatti, and P. L. Luisi, J. Biochem and Biphys Meth. 12, 325 (1986). PA0 (10) P. L. Luisi, Angew Chem Int Ed Engl. 24, 439 (1985). PA0 (11) K. Larsson, P. Adlecreutz, and B. Mattiasson, "Biocatalysis in Organic Media," C Laane, J. Tramper, and M. D. Lilly, Eds., Elsevier Science Publishers B. V., Amsterdam, p. 355 (1986). PA0 (12) P. L. Luisi, P. Luthi, I. Tomka, J. Prenosil, and A. Pande, Annals N.Y. Acad. of Sci. 434, 549 (1984). PA0 (13) D. Han and J. S. Rhee, Biotech and Bioeng 28, 1250 (1986). PA0 (14) M. A. Hernandez-Torres, J. S. Landy, and J. G. Dorsey, Anal. Chem. 58,744 (1986). PA0 (15) R. W. Gale, J. L. Fulton, and R. D. Smith, Anal Cheml 59, 1977 (1987). PA0 (16) K. E. Goklen and T. Alan Hatton, Biotech Progress 1, 69, (1985). PA0 (17) L. J. Karr, S. G. Shafer, J. M. Harris, J. M. Van Alstine, and R. S. Snyder, J. Chromatography 354, 269 (1986). PA0 (18) A. N. Eremin, M. I. Savenkova, and D. I. Metelitsa, Bioorg Khim 12, 606 (1986).